Fluorescence Labeling of Circulating Tumor Cells with a Folate Receptor-Targeted Molecular Probe for DiffuseIn VivoFlow Cytometry

Purpose We recently developed a new instrument called diffusein vivoflow cytometry (DiFC) for enumeration of rare fluorescently labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins or were pre-labeled with a fluorescent dyeex vivo. In this work, we investigated the use of a folate receptor (FR)-targeted fluorescence molecular probe forin vivolabeling of FR+ CTCs for DiFC. Procedures We used EC-17, a FITC-folic acid conjugate that has been used in clinical trials for fluorescence-guided surgery. We studied the affinity of EC-17 for FR+ L1210A and KB cancer cells. We also tested FR- MM.1S cells. We tested the labeling specificity in cells in culturein vitroand in whole blood. We also studied the detectability of labeled cells in micein vivowith DiFC. Results EC-17 showed a high affinity for FR+ L1210A and KB cellsin vitro. In whole blood, 85.4 % of L1210A and 80.9 % of KB cells were labeled above non-specific background with EC-17, and negligible binding to FR- MM.1S cells was observed. In addition, EC-17-labeled CTCs were readily detectable in circulation in mice with DiFC. Conclusions This work demonstrates the feasibility of labeling CTCs with a cell-surface receptor-targeted probe for DiFC, greatly expanding the potential utility of the method for pre-clinical animal models. Because DiFC uses diffuse light, this method could be also used to enumerate CTCs in larger animal models and potentially even in humans.